Oligosaccharides comprising an aminooxy group and conjugates thereof

ABSTRACT

The invention provides methods for the synthesis of oligosaccharides comprising an aminooxy group. The invention further provides oligosaccharides comprising an aminooxy group, methods for coupling oligosaccharides comprising an aminooxy group to glycoproteins, and oligosaccharide-protein conjugates. Also provided are methods of treating a lysosomal storage disorder in a mammal by administration of an oligosaccharide-protein conjugate.

This application claims the benefit of U.S. Provisional Application No.60/885,471, filed Jan. 18, 2007, the disclosure of which is incorporatedherein by reference.

The invention relates generally to methods for the synthesis ofoligosaccharides comprising an aminooxy group from oligosaccharidescomprising a reactive group. In another embodiment, the inventionfurther relates to oligosaccharides comprising an aminooxy group. Theinvention also relates to methods of conjugating oligosaccharidescomprising an aminooxy group to proteins, including glycoproteins (suchas, e.g., lysosomal enzymes), and to compositions ofoligosaccharide-protein conjugates, includingoligosaccharide-glycoprotein conjugates. Another embodiment of theinvention relates to methods of treating lysosomal storage disordersusing such oligosaccharide-lysosomal enzyme conjugates.

Lysosomal storage disorders (LSDs) are a class of rare metabolicdisorders comprising over forty genetic diseases involving a deficiencyin the activity of lysosomal hydrolases. A hallmark feature of LSDs isthe abnormal accumulation of lysosomal metabolites, which leads to theformation of large numbers of distended lysosomes.

LSDs can be treated by administration of the active version of theenzyme deficient in the patient, a process termed enzyme replacementtherapy (ERT). The administered replacement enzyme bearing a terminalmannose-6-phosphate (M6P) is taken up by target cells throughcell-surface-associated cation-independent M6P receptor(CI-MPR)-mediated endocytosis, and directed to the lysosome.

In general, poorly phosphorylated replacement enzymes are notinternalized by the M6P receptor on cell surfaces, and therefore cannotbe directed to the lysosome where they function. Consequently, a lowdegree of mannose phosphorylation can have a significant and deleteriouseffect on the therapeutic efficacy of a replacement enzyme.

Methods thus have been developed for increasing the M6P content ofreplacement enzymes. U.S. Pat. No. 7,001,994, for example, describes amethod for coupling oligosaccharides comprising M6P with glycoproteins.The oligosaccharides of the glycoproteins are first oxidized withperiodate or galactose oxidase to result in the formation of carbonylgroups, which are then chemically conjugated with an oligosaccharidefunctionalized at the reducing end with a carbonyl-reactive group (suchas, e.g., a hydrazine, hydrazide, aminooxy, thiosemicarbazide,semicarbazide, or amine group) to yield an oligosaccharide-glycoproteinconjugate.

A conjugate of the lysosomal enzyme acid α-glucosidase (GAA) with abis-M6P oligosaccharide was prepared by the above-described method, andfound to be more effective in reducing skeletal and cardiac muscleglycogen than recombinant human GAA in a murine model of Pompe disease,an autosomal recessive muscular disease resulting from a metabolicdeficiency of GAA, and characterized by the accumulation of lysosomalglycogen.

Aminooxy groups are particularly useful carbonyl-reactive groups for theconjugation reactions described above, as the resulting conjugatescomprise a relatively stable oxime linkage. Therefore, there is a needfor methods for the preparation of aminooxy functionalizedoligosaccharides.

The present invention provides methods of preparing oligosaccharidescomprising an aminooxy group. These methods are generally applicable toa broad range of protected and unprotected oligosaccharides, such as,e.g., branched and unbranched, and phosphorylated and unphosphorylated,oligosaccharides. In certain embodiments, the oligosaccharide may be adisaccharide, trisaccharide, tetrasaccharide, pentasaccharide,hexasaccharide, heptasaccharide, or greater. The oligosaccharide may, incertain embodiments, comprise at least one M6P residue. In someembodiments, the oligosaccharide may comprise at least 1, 2, 3, 4, 5, 6,or 7 terminal M6P residues.

The invention provides a method of preparing an oligosaccharidecomprising an aminooxy group from an oligosaccharide comprising areactive group. The method comprises:

-   -   (a) providing an oligosaccharide comprising a first reactive        group;    -   (b) providing an aminooxy compound comprising an aminooxy group        and a second reactive group; and    -   (c) reacting the first reactive group of the oligosaccharide        with the second reactive group of the aminooxy compound, thereby        preparing the oligosaccharide comprising an aminooxy group.

The first and second reactive groups may be chosen from, e.g.,hydrazine, hydrazide, thiosemicarbazide, semicarbazide, amine, carboxyl,activated ester, acyl halide, acyl azide, alkyl halide, anhydride,isothiocyanate, isocyanate, and sulfonyl halide groups.

In some embodiments, the aminooxy compound is chosen from compounds ofFormula II:

wherein Y is the second reactive group, Z is chosen from alkyl, alkenyl,alkynyl, aryl, heteroaryl, and heterocyclyl, and P is chosen from aminoprotecting groups (such as, e.g., carbamate protecting groups). Forexample, in some embodiments, Y may be a carboxyl, activated ester, acylhalide (such as, e.g., an acyl fluoride or acyl chloride), acyl azide,alkyl halide, anhydride, isothiocyanate, isocyanate, or sulfonyl halide(such as, e.g., a sulfonyl chloride or sulfonyl bromide). In otherembodiments, Y may be, e.g., a hydrazine, hydrazide, thiosemicarbazide,semicarbazide, or amine group.

In certain embodiments, the aminooxy compound of Formula II is chosenfrom compounds of Formula III:

wherein Y is the second reactive group, n is chosen from integersranging from 1 to 10, and P is chosen from amino protecting groups.

In certain embodiments, the aminooxy compound comprises an aminoprotecting group, and the method further comprises a step (d),deprotecting the oligosaccharide comprising an aminooxy group.

The invention further provides an oligosaccharide comprising (1) anaminooxy group and (2) mannose-6-phosphate. In some embodiments, thatoligosaccharide is prepared by the methods described above. For example,in some embodiments, the invention provides an oligosaccharidecomprising an aminooxy group of Formula IV:

wherein m and p are independently chosen from integers ranging from 1 to10.

In another embodiment, the invention provides an oligosaccharide ofFormula V:

In another embodiment, the invention provides methods of coupling anoligosaccharide to a protein. In one embodiment, the method comprises:

-   -   (a) providing an oligosaccharide comprising an aminooxy group;    -   (b) providing a protein having at least one carbonyl group; and    -   (c) reacting the aminooxy group of the oligosaccharide with the        at least one carbonyl group of the protein,        thereby coupling the oligosaccharide to the protein.

In other embodiments, the invention further provides anoligosaccharide-protein conjugate comprising (1) a protein, (2) anoligosaccharide, and (3) an oxime group connecting the protein and theoligosaccharide. For example, in some embodiments, the inventionprovides an oligosaccharide-protein conjugate prepared by the methodsdisclosed above. In certain embodiments, the oligosaccharide-proteinconjugate is an oligosaccharide-glycoprotein conjugate. In certainembodiments, the oligosaccharide-glycoprotein conjugate is the conjugateof an oligosaccharide comprising at least one M6P and of a lysosomalenzyme such as, e.g., a lysosomal hydrolase. In some embodiments, theinvention provides pharmaceutical compositions comprising anoligosaccharide-protein conjugate of the invention.

Another embodiment of the invention provides methods of treating alysosomal storage disorder such as, e.g., those disclosed in Table 1. Insome embodiments, the methods comprise administering to a mammal anoligosaccharide-glycoprotein conjugate of the invention, wherein theoligosaccharide comprises at least one M6P and the glycoprotein is alysosomal hydrolase. This disclosure further provides the use of aconjugate of the invention for treating a lysosomal storage disorder ina subject in need thereof, and in the manufacture of a medicament fortreating a lysosomal storage disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a reaction scheme depicting an illustrative embodiment of themethods of the invention. Oligosaccharide 1, having a first reactivegroup (a hydrazide group), is reacted with aminooxy compound 2 inpresence of the catalyst 3-hydroxy-1,2,3-benzotriazin-4(3H)-one(DHBt-OH), to yield oligosaccharide 3. The tert-butyloxycarbonyl (t-Boc)amino protecting group of oligosaccharide 3 is then removed with 50%trifluoroacetic acid/dichloromethane (TFA/DCM) to yield oligosaccharide4.

FIG. 2 depicts a series of gel chromatographs of intermediates in thesynthetic scheme described in FIG. 1. FIG. 2A is a Dionex analyticalchromatograph of starting oligosaccharide 1. FIG. 2B is a Dionexanalytical chromatograph of oligosaccharide 3. FIG. 2C is a Dionexanalytical chromatograph of oligosaccharide 4.

FIG. 3A is a mass spectrum of oligosaccharide 1 (calculated molecularweight=1250; calculated molecular weight of sodium salt=1338). FIG. 3Bis a mass spectrum of oligosaccharide 4 (calculated molecularweight=1323; calculated molecular weight of sodium salt=1411).

FIG. 4 is a reaction scheme depicting an illustrative embodiment of themethods of the invention. Oligosaccharide 5 having a first reactivegroup (a carboxyl group) is reacted with aminooxy compound 6 in presenceof the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) and the catalyst N-hydroxysuccinimide (NHS), to yieldaminooxy-containing oligosaccharide 3. The Boc amino protecting group ofoligosaccharide 3 is then removed with 50% TFA/DCM to yieldoligosaccharide 4.

I. PREPARATION OF AN OLIGOSACCHARIDE COMPRISING AN AMINOOXY GROUP A.Oligosaccharide Comprising a Reactive Group

The methods of the invention are applicable to a broad range ofoligosaccharides comprising a reactive group. As used herein, anoligosaccharide refers to a disaccharide, trisaccharide,tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, orlarger oligosaccharide (such as, e.g., an oligosaccharide comprising2-50, 2-10, 8-25, or 8-50 saccharide units). Accordingly, in variousembodiments, an oligosaccharide may be, e.g., a disaccharide,trisaccharide, tetrasaccharide, a pentasaccharide, a hexasaccharide, aheptasaccharide, or a larger oligosaccharide. An oligosaccharide may bemono-, bi-, tri-, tetra-, or penta-antennary in structure. Anoligosaccharide may comprise 0, 1, 2, 3, 4, or more branch points.

The reactive group on the oligosaccharide, also referred to as a firstreactive group, may be, in some embodiments, e.g., a hydrazine group,hydrazide group, semicarbazide group, thiosemicarbazide, or amine group.In some embodiments, the first reactive group may be, e.g., a carboxyl,ester (such as, e.g., an activated ester), acyl halide (such as, e.g.,acyl fluoride or acyl chloride), acyl azide, alkyl halide, anhydride,isothiocyanate, isocyanate, or sulfonyl halide (such as, e.g., sulfonylchloride or sulfonyl bromide) group.

The first reactive group may be connected to the reducing end of theoligosaccharide or may be located anywhere in the oligosaccharide. Thefirst reactive group may, in certain embodiments, be connected throughone or more linkers to the oligosaccharide. A linker, as used herein,may be chosen from, e.g., a combination of optionally substituted alkyl,alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy,aryloxy, and heterocyclyloxy groups. A linker may be interrupted orterminated by one or more heteroatoms such as, e.g., nitrogen, sulfur,and oxygen. For example, a linker, in some embodiments, may comprise oneor more ether, ester, or amide group.

Any chemical group of the linker (such as, e.g., alkyl, alkenyl,alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy, alkoxy, aryloxy, andheterocyclyloxy) may be substituted or unsubstituted, unless otherwisestated. Substituents may be chosen from, e.g., acyl, acylamino, acyloxy,alkenyl, alkoxy, alkyl, alkynyl, amido, amino, aryl, aryloxy, azido,carbamoyl, carboalkoxy, carboxy, cyano, cycloalkyl, formyl, guanidino,halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, nitro, oxo,phosphonamino, sulfinyl, sulfonamino, sulfonate, sulfonyl, thio,thioacylamino, thioureido, and ureido. The substituents may themselvesbe substituted or unsubstituted, and may be interrupted or terminated byone or more heteroatoms such as, e.g., nitrogen, sulfur, and oxygen.

In certain embodiments, an oligosaccharide may comprise at least oneprotecting group. The term “protecting group” refers to any substituentthat may be used to prevent a functional group (such as, e.g., an aminegroup, a carboxyl group, a hydroxyl group, a hydrazine group, ahydrazide group, a semicarbazide group, or a thiosemicarbazide group) ona molecule from undergoing a chemical reaction while chemical changeoccurs elsewhere in the molecule. A protecting group can be removedunder the appropriate chemical conditions. Numerous protecting groupsare known to those skilled in the art, and examples of protectinggroups, methods for their addition, and methods for their removal can befound in, e.g., Greene et al., Protective Groups in Organic Synthesis,3^(rd) ed., John Wiley and Sons: New York, 1999 and Kocienski,Protecting Groups, 3^(rd) ed., Georg Thieme Verlag: Stuttgard, Germany,2005, the disclosures of which are herein incorporated by reference. Incertain embodiments, the oligosaccharide may comprise at least oneprotecting group chosen from hydroxyl protecting groups, carboxylprotecting groups, and amino protecting groups. In other embodiments, anoligosaccharide may be “unprotected,” and may not comprise anyprotecting groups.

An oligosaccharide may be isolated from a natural source or may beprepared by chemical or enzymatic synthesis. An oligosaccharide isolatedfrom a natural source may be homogeneous or may be a heterogeneousmixture of related oligosaccharides. In some embodiments, anoligosaccharide may be prepared by chemical or enzymatic modification ofan oligosaccharide isolated from a natural source (“semi-synthesis”). Insome embodiments, the oligosaccharide may be a synthetic oligosaccharidehaving the chemical structure of a naturally occurring oligosaccharide.

In some embodiments, an oligosaccharide may comprise a monosaccharidethat is recognized by a particular receptor. The monosacchariderecognized by a particular receptor may be chosen from, e.g., galactose,GalNAc, mannose, M6P, glucose, GlcNAc, sialic acid, or sulfated sialicacid residue. An oligosaccharide may, in certain embodiments, compriseat least one M6P residue, such as, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 M6P residues.

The monosaccharide recognized by a particular receptor may be, in someembodiments, a penultimate monosaccharide or a terminal monosaccharide.In some embodiments, the monosaccharide recognized by a particularreceptor may be a terminal galactose, mannose, M6P, glucose, GlcNAc, orsialic acid residue. An oligosaccharide may, in some embodiments,contain at least 1, 2, 3, 4, 5, 6, 7 terminal M6P residues.

In certain embodiments, the oligosaccharide comprising a reactive groupmay be an M6P-containing hexasaccharide of Formula Ia:

The oligosaccharide of Formula Ia can be described asbutyrylhydrazine-4-yl6-O-phosphono-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)-α-D-mannopyranosyl-(1→6)-[6-O-phosphono-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranoside.

In certain embodiments, the oligosaccharide comprising a reactive groupmay be an M6P-containing hexasaccharide of Formula Ib:

B. Aminooxy Compound

As used herein, an aminooxy compound may be any compound comprising anaminooxy group and a second reactive group, wherein the second reactivegroup may react with a first reactive group on an oligosaccharide toform a covalent bond. For example, in some embodiments, the secondreactive group may be a carboxyl, ester (such as, e.g., an activatedester), acyl halide (such as, e.g., an acyl fluoride or acyl chloride),acyl azide, anhydride, isothiocyanate, isocyanate, or sulfonyl halide(such as, e.g., a sulfonyl chloride or sulfonyl bromide) group. In otherembodiments, the second reactive group may be, e.g., a hydrazine group,hydrazide group, semicarbazide group, thiosemicarbazide, or amine group.

In certain embodiments, the nitrogen of the aminooxy group of theaminooxy compound is protected with an amino protecting group. Numerousamino protecting groups are known to those skilled in the art, andexamples of amino protecting groups, methods for their addition, andmethods for their removal can be found in pp. 494-653 of Greene et al.,Protective Groups in Organic Synthesis, 3^(rd) ed., John Wiley and Sons:New York, 1999; Chapter 8 of Kocienski, Protecting Groups, 3^(rd) ed.,Georg Thieme Verlag: Stuttgard, Germany, 2005; Bodanszky, Principles ofPeptide Synthesis, Springer Verlag: New York, 1993; Lloyd-Williams etal., Chemical Approaches to the Synthesis of Peptides and Proteins, CRCPress: Boca Raton, Fla., 1997; and Stewart et al., Solid Phase PeptideSynthesis, 2nd ed., Pierce Chemical Co.: Rockford, Ill., 1984, theinventions of which are incorporated herein by reference.

In some embodiments, the aminooxy compound is chosen from compounds ofFormula II:

wherein Y is the second reactive group, Z is chosen from alkyl, alkenyl,alkynyl, heteroaryl, aryl, and heterocyclyl, and P is chosen from aminoprotecting groups.

As used herein, any chemical group on the aminooxy compound (such as,e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyloxy,alkoxy, aryloxy, and heterocyclyloxy) may be substituted orunsubstituted, and may be interrupted by one or more chemical groups,unless otherwise stated. Substituents and interrupting chemical groupsmay be chosen from, e.g., acyl, acylamino, acyloxy, alkenyl, alkoxy,alkyl, alkynyl, amido, amino, aryl, aryloxy, azido, carbamoyl,carboalkoxy, carboxy, cyano, cycloalkyl, formyl, guanidino, halo,heteroaryl, heterocyclyl, hydroxy, iminoamino, nitro, oxo,phosphonamino, sulfinyl, sulfonamino, sulfonate, sulfonyl, thio,thioacylamino, thioureido, and ureido. The substituents may themselvesbe substituted or unsubstituted, and may be interrupted or terminated byone or more heteroatoms such as, e.g., nitrogen, sulfur, and oxygen.

In certain embodiments, Y may be chosen from, for example:

-   -   wherein X is chosen from halogens, azide, acyloxy, alkoxy,        aryloxy, heteroaryloxy, and heterocyclyloxy.

In certain embodiments, the aminooxy compound is an activated ester. Asused herein, an activated ester is an ester that reacts to form an amidebond under mild conditions. In general, an activated ester is an esterof a relatively acidic alcohol. In certain embodiments, the aminooxycompound of Formula II is an activated ester of formula

and X is chosen from alkoxy, aryloxy, heteroaryloxy, andheterocyclyloxy. For example, X may be chosen from:

In other embodiments, Y is chosen from, e.g., hydrazide, hydrazine,thiosemicarbazide, semicarbazide, and amine groups.

In some embodiments, Z may comprise, for example, a carbonyl, ether,ester, or amide group. In some embodiments, Z may be, for example, alkylinterrupted by one or more heteroatoms, such as an oligoethyleneglycol.For example, Z may be monoethyleneglycol, diethyleneglycol,triethyleneglycol, tetraethyleneglycol, or larger oligoethyleneglycol.

In some embodiments, Z may be, for example, alkyl substituted with oxoand interrupted by one or more heteroatoms, such as an oligopeptide. Forexample, the oligopeptide may comprise one, two, three, four, five, six,or more component amino acids. The amino acids may be, for example,α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and co-aminoacids. An amino acid may have R or S chirality at any chiral atom. Anamino acid may be chosen from, e.g., alanine, β-alanine, α-aminoadipicacid, 2-aminobutanoic acid, 4-aminobutanoic acid,1-aminocyclopentanecarboxylic acid, 6-aminohexanoic acid,2-aminoheptanedioic acid, 7-aminoheptanoic acid, 2-aminoisobutyric acid,aminomethylpyrrole carboxylic acid, 8-amino-3,6-dioxa-octanoic acid,aminopiperidinecarboxylic acid, 3-amino-propionic acid, aminoserine,aminotetrahydropyran-4-carboxylic acid, arginine, asparagine, asparticacid, azetidine carboxylic acid, benzothiazolylalanine, butylglycine,carnitine, 4-chlorophenylalanine, citrulline, cyclohexylalanine,cyclohexylstatine, cysteine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid, dihydroxyphenylalanine, dimethylthiazolidinecarboxylic acid, glutamic acid, glutamine, glycine, histidine,homoserine, hydroxyproline, isoleucine, isonipecotic acid, leucine,lysine, methanoproline, methionine, norleucine, norvaline, ornithine,p-aminobenzoic acid, penicillamine, phenylalanine, phenyiglycine, piperidinylalanine, piperidinylglycine, proline, pyrrolidinylalanine,sarcosine, selenocysteine, serine, statine, tetrahydropyranglycine,thienylalanine, threonine, tryptophan, tyrosine, valine,allo-isoleucine, allo-threonine, 2,6-diamino-4-hexanoic acid,2,6-diaminopimelic acid, 2,3-diaminopropionic acid, dicarboxidine,homoarginine, homocitrulline, homocysteine, homocystine,homophenylalanine, homoproline, and 4-hydrazinobenzoic acid.

P may be chosen from amino protecting groups known to those of skill inthe art. In some embodiments, P may be a carbamate protecting group,such as, e.g., a (9-fluorenylmethyl)carbamate (Fmoc),(tert-butyloxy)carbamate (t-Boc), (trichloroethyl)carbamate (Troc), orallylcarbamate (Alloc) protecting group. In other embodiments, P may bea non-carbamate protecting group, such as, e.g., an amide protectinggroup such as a phthalimide or a trifluoroacetamide protecting group.

In some embodiments, the aminooxy compound of Formula II is chosen fromcompounds of Formula III:

wherein Y and P are as disclosed above, and n is chosen from integersranging from 1 to 10.

In certain embodiments, n may be chosen from integers from the followingranges: 1-4, 2-6, 2-8, 3-6, and 4-10. In illustrative embodiments, n is1.

In one illustrative embodiment, the aminooxy compound is t-Boc-aminooxyacetic acid tetrafluorophenyl ester, the structure of which is depictedbelow.

In another illustrative embodiment, the aminooxy compound has thestructure depicted below.

C. Methods of Preparing an Oligosaccharide Comprising an Aminooxy Group

In another embodiment, the invention provides a method of preparing anoligosaccharide comprising an aminooxy group from an oligosaccharidecomprising a reactive group. The method comprises:

-   -   (a) providing an oligosaccharide comprising a first reactive        group;    -   (b) providing an aminooxy compound comprising a second reactive        group; and    -   (c) reacting the first reactive group of the oligosaccharide        with the second reactive group of the aminooxy compound,        thereby preparing the oligosaccharide comprising an aminooxy        group.

The oligosaccharide comprising a first reactive compound may be, e.g.,any oligosaccharide comprising a reactive group as described supra. Inillustrative embodiments, the oligosaccharide comprising a firstreactive group is an oligosaccharide of Formula Ia or an oligosaccharideof Formula Ib. The aminooxy compound comprising a second reactive groupmay be any aminooxy compound comprising a reactive group, as describedsupra.

The terms “first reactive group” and “second reactive group,” as usedherein, do not denote any particular experimental sequence. I.e., step(c), reacting the first reactive group of the oligosaccharide with thesecond reactive group of the aminooxy compound, may be accomplished byany order of addition of the reactants. For example, the oligosaccharidecomprising a first reactive group may be added to the aminooxy compoundcomprising the second reactive group, or vice versa. In another example,both the oligosaccharide and the aminooxy compound may be addedsimultaneously to a reaction vessel.

Step (c) may occur under any suitable conditions (e.g., solvent andtemperature) known to those of ordinary skill in the art. In certainembodiments, one or more additional reagents, such as, e.g., couplingreagents and catalysts, may be present during step (c). A couplingreagent, as used herein, is a reagent that may be used to form acovalent bond between the first reactive group and the second reactivegroup.

In some embodiments, such as, e.g., when the first or second reactivegroup is a carboxyl group, the reaction conditions may comprise acoupling reagent. Coupling reagents may be chosen from, e.g.,phosphonium coupling reagents such as, e.g., BOP(benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate), PyBOP®(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), and PyBroP® (bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate), and from aminium (uronium) coupling reagents suchas, e.g., HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), HATU(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate). Coupling reagents may also be chosen from, e.g.,carbodiimide coupling reagents such as, e.g., DIC(1,3-diisopropylcarbodiimide), CDI (1,1′ carbonyl diimidazole), and EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). For example, in someillustrative embodiments, the coupling reagent is EDC. In certainembodiments, the reaction conditions comprise both a coupling reagentand a catalyst.

The reaction conditions may, in certain embodiments, comprise acatalyst. The catalyst may be chosen from any suitable catalyst known tothose of skill in the art, such as, e.g., DHBt-OH(3-hydroxy-1,2,3-benzotriazin-4(3H)-one), HOBt (N-hydroxybenzotriazole),DMAP (4-dimethylaminopyridine), NHS (N-hydroxysuccinimide),N-hydroxysulfosuccinimide, HONB(N-hydroxy-5-norbornene-endo-2,3-dicarboximide), or a tetrabutylammoniumsalt such as, e.g., TBAI (tetrabutylammonium iodide). In someillustrative embodiments, the reaction conditions comprise the catalystDHBt-OH or the catalyst NHS.

In some embodiments, step (c), reacting the first reactive group of theoligosaccharide with the second reactive group of the aminooxy compoundresults in the formation of an amide bond. Conditions suitable for theformation of an amide bond are well known to those of ordinary skill inthe art, and are described in, e.g., Chan et al., eds., Fmoc Solid PhasePeptide Synthesis: A Practical Approach, Oxford University Press: NewYork, 2000; Bodanszky, Principles of Peptide Synthesis, Springer Verlag:New York, 1993; Lloyd-Williams et al., Chemical Approaches to theSynthesis of Peptides and Proteins, CRC Press: Boca Raton, Fla., 1997;and the Novabiochem® (San Diego, Calif.) Catalog.

In certain embodiments, the aminooxy compound comprises an aminoprotecting group, and the method comprises a further step (d),deprotecting the oligosaccharide comprising an aminooxy group to removethe amino protecting group. Deprotection may occur under any suitableconditions known to those of skill in the art, such as, e.g., thosetaught in pp. 494-653 of Greene et al., Protective Groups in OrganicSynthesis, 3^(rd) ed., John Wiley and Sons: New York, 1999 andKocienski, Protecting Groups, 3^(rd) ed., Georg Thieme Verlag:Stuttgard, Germany, 2005, the inventions of which are incorporatedherein by reference.

An illustrative embodiment of the method of the invention provides amethod of preparing an M6P-containing oligosaccharide comprising anaminooxy group. The method comprises:

-   -   (a) providing an oligosaccharide comprising a first reactive        group, wherein the oligosaccharide is

-   -   (b) providing an aminooxy compound comprising a second reactive        group, wherein the aminooxy compound is chosen from compounds of        Formula III:

-   -   -   wherein n is chosen from integers ranging from 1 to 10, P is            chosen from amino protecting groups, and Y is a second            reactive group; and

    -   (c) reacting the first reactive group of the oligosaccharide        with the second reactive group of the aminooxy compound,        thereby preparing the oligosaccharide comprising an aminooxy        group.

In certain embodiments, Y in Formula III is

where X is chosen from hydroxy, aryloxy, heteroaryloxy, andheterocyclyloxy. For example, in certain illustrative embodiments, X is

In illustrative embodiments, the aminooxy compound is

In certain embodiments, the first reactive group of the oligosaccharidemay be reacted with the second reactive group of the aminooxy compoundin the presence of a coupling agent, such as, e.g., EDC, and/or acatalyst, such as, e.g., DHBt-OH.

Another illustrative embodiment of the method of the inventioncomprises:

-   -   (a) providing an oligosaccharide comprising a first reactive        group, wherein the oligosaccharide is

-   -   (b) providing an aminooxy compound comprising a second reactive        group, wherein the aminooxy compound is chosen from compounds of        Formula III:

-   -   -   wherein n is chosen from integers ranging from 1 to 10, P is            chosen from amino protecting groups, and Y is a second            reactive group; and

    -   (c) reacting the first reactive group of the oligosaccharide        with the second reactive group of the aminooxy compound,        thereby preparing the oligosaccharide comprising an aminooxy        group.

In certain embodiments, Y in Formula III is a hydrazine, hydrazide,aminooxy, thiosemicarbazide, semicarbazide, or amine group. In certainembodiments, Y in Formula III is

In illustrative embodiments, the aminooxy compound is

In certain embodiments, the first reactive group of the oligosaccharidemay be reacted with the second reactive group of the aminooxy compoundin the presence of a coupling agent, such as, e.g., EDC, and/or acatalyst, such as, e.g., NHS.

II. OLIGOSACCHARIDES COMPRISING AN AMINOOXY GROUP

The present invention also provides oligosaccharides comprising anaminooxy group. In some embodiments, the invention providesoligosaccharides comprising an aminooxy group prepared by the methodsdisclosed above. The oligosaccharide comprising an aminooxy group maycomprise, for example, at least 2, 3, 4, 5, 6, or more monosaccharides,including, e.g., at least one galactose, GalNAc, mannose, M6P, glucose,GlcNAc, sialic acid, or sulfated sialic acid residue. Such anoligosaccharide may be mono-, bi-, tri-, tetra-, or penta-antennary instructure, and may contain 0, 1, 2, 3, 4, or more branch points.

In some embodiments, the present invention provides an oligosaccharidecomprising (1) an aminooxy group and (2) mannose-6-phosphate. Theoligosaccharide comprising an aminooxy group may, in some embodiments,comprise, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 M6P residues. In someembodiments, the oligosaccharide comprising an aminooxy group maycomprise at least 1, 2, 3, 4, or more terminal or penultimate M6Presidues.

The oligosaccharides comprising an aminooxy group are, in certainembodiments, chosen from oligosaccharides of Formula IV:

wherein m and p are independently chosen from integers ranging from 1 to10. For example, in certain embodiments, m and p may be independentlychosen from integers selected from the following ranges: 1-4, 2-6, 2-8,3-6, and 4-10. In illustrative embodiments, m is 3 and p is 1.

In other embodiments, the aminooxy group is directly linked to thereducing end of the oligosaccharide. For example, in some embodiments,the oligosaccharide comprising an aminooxy group may be anoligosaccharide of Formula V:

III. CONJUGATION OF AN OLIGOSACCHARIDE COMPRISING AN AMINOOXY GROUP WITHA PROTEIN A. Oligosaccharide

The oligosaccharide to be conjugated with a protein may be chosen fromany oligosaccharide comprising a reactive group, as discussed supra, andfrom any oligosaccharide comprising an aminooxy group, as discussedsupra. For example, in some embodiments, the oligosaccharide to beconjugated may be an oligosaccharide of Formula Ia, Formula Ib, FormulaIV or Formula V.

B. Protein

The conjugation methods described herein are broadly applicable to anypure protein, partially purified protein, or fragment thereof, having atleast one carbonyl group (where a carbonyl group is a ketone or analdehyde), including isolated proteins and recombinantly orsynthetically produced proteins. The terms “pure,” “purified,” and“isolated” refer to a molecule that is substantially free of its naturalenvironment. For instance, a pure protein is substantially free ofcellular material and/or other proteins from the cell or tissue sourcefrom which it is derived. The term refers to preparations that are, forexample, at least 70% to 80%, 80% to 90%, 90 to 95%; or at least 95%,96%, 97%, 98%, 99%, or 100% (w/w) pure.

In other embodiments, the protein may be an enzyme that has optimalactivity, as measured by an activity assay, at a pH ranging from 1-7,such as, e.g., 1-3, 2-5, 3-6, 4-5, 5-6, or 4-6. For example, the enzymemay have a pH optimum at a pH ranging from 4-6.

In some embodiments, the protein may be an enzyme that has anisoelectric point (pI), ranging from 1 to 8, such as, e.g., from 1-3,2-5, 3-8, 4-5, 5-6, 4-6, 5-8, 6-8, or 7-8. The pI of a protein may bemay be measured using, e.g., isoelectric focusing gel electrophoresis.

In some embodiments, the protein containing a carbonyl group is obtainedby the use of an expression system having an expanded genetic code, asdescribed in, e.g., Wang et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003). In such a case, the carbonyl group may be located on amino acidside chain, as translated.

In certain embodiments, the protein having at least one carbonyl groupis a protein having at least one oligosaccharide (i.e., a glycoprotein).For example, a glycoprotein having at least one carbonyl group may beobtained by oxidation of that glycoprotein by any means known to thoseof skill in the art. In some embodiments, e.g., a glycoprotein having atleast one carbonyl group may be obtained by oxidation of thatglycoprotein with periodate (e.g., sodium periodate) or with galactoseoxidase. In such a case, the carbonyl group may be located at a proteinglycosylation site.

In certain embodiments, the protein having at least one carbonyl groupis a glycoprotein, such as a therapeutic glycoprotein. A therapeuticglycoprotein may be targeted to the lysosome by conjugation with anoligosaccharide comprising mannose-6-phosphate. For example, theglycoprotein may be a lysosomal enzyme, including an ERT enzyme. Theenzyme may be a lysosomal hydrolase, including those listed in Table 1.In certain embodiments, the lyosomal hydrolase is chosen from, e.g.,α-glucosidase, α-galactosidase A, and acid sphingomyelinase. In certainembodiments, the lyosomal hydrolase is GAA.

TABLE 1 Examples of LSDs and Corresponding Lysosomal HydrolasesLysosomal Storage Disorder Defective Enzyme Fabry α-Galactosidase AFarber Acid ceramidase Fucosidosis Acid α-L-fucosidase Gaucher types 1,2, and 3 Acid β-glucosidase G_(M1) gangliosidosis Acid β-galactosidaseHunter (Mucopolysaccharidosis Iduronate-2-sulfatase (MPS) II)Hurler-Scheie, Hurler, Scheie α-L-Iduronidase (MPS I) KrabbeGalactocerebrosidase α-Mannosidosis Acid α-mannosidase β-MannosidosisAcid β-mannosidase Maroteaux-Lamy (MPS VI) Arylsulfatase B Metachromaticleukodystrophy Arylsulfatase A Morquio A (MPS IV)N-Acetylgalactosamine-6-sulfate sulfatase Morquio B (MPS IV) Acidβ-galactosidase Niemann-Pick A and B Acid sphingomyelinase (ASM) PompeAcid α-glucosidase (α-glucosidase; GAA) Sandhoff β-Hexosaminidase BSanfilippo A (MPS III) Heparan N-sulfatase Sanfilippo B (MPS III)α-N-Acetylglucosaminidase Sanfilippo C (MPS III)Acetyl-CoA:α-glucosaminide N-acetyltransferase Sanfilippo D (MPS III)N-Acetylglucosamine-6-sulfate sulfatase Schindler-Kanzakiα-N-acetylgalactosaminidase Sialidosis Sialidase Sly (MPS VII)β-Glucuronidase Tay-Sachs β-Hexosaminidase A

In certain embodiments, the glycoprotein may be a glycoprotein having atleast 1, 2, 3, 4, 5, or more N-linked or O-linked glycosylated aminoacid residues. In other embodiments, the protein may have 1, 2, 3, 4, 5or more consensus sites for N-linked or O-linked glycosylation, at leastone of which is glycosylated.

In certain embodiments, the protein may be a ligand for a receptor. Forexample, in some embodiments the protein may be a glycoprotein thatbinds to a receptor that recognizes a sugar such as, e.g., mannose ormannose-6-phosphate. In some embodiments, the glycoprotein may bind to,e.g., the asialoglycoprotein receptor, the cation-dependentmannose-6-phosphate receptor, the insulin-like growth factorII/cation-independent mannose-6-phosphate receptor, or the macrophagemannose receptor.

In certain embodiments, the protein is a glycoprotein that, whenconjugated to an oligosaccharide comprising mannose-6-phosphate, isinternalized more efficiently by a target cell (e.g., viaCI-MPR-mediated endocytosis) than is the corresponding unconjugatedglycoprotein. For example, the conjugated glycoprotein may beinternalized more efficiently than the unconjugated glycoprotein by,e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or90% (w/w) in a given time period. In other embodiments, at least 2, 3,4, 5, 6, 7, 8, 9, or 10 fold (w/w) as much of the conjugatedglycoprotein may be internalized, relative to the unconjugatedglycoprotein, in a given time period. The referenced time period may be,for example, 10, 30, 45 minutes or 1, 2, 3, 5, 6, 12, 24, 48, or 72hours, or more.

C. Methods of Coupling an Oligosaccharide to a Protein

The invention provides methods of coupling an oligosaccharide to aprotein, such as, e.g., a glycoprotein. In one embodiment, the methodcomprises:

-   -   (a) providing an oligosaccharide comprising an aminooxy group;    -   (b) providing a protein having at least one carbonyl group; and    -   (c) reacting the aminooxy group of the oligosaccharide with the        at least one carbonyl group of the protein,        thereby coupling the oligosaccharide to the protein.

In certain embodiments, the methods further comprise adding a reducingagent to the coupled lysosomal enzyme. The reducing agent may be anyreducing agent known to those of skill in the art, such as, e.g., sodiumcyanoborohydride or sodium triacetoxyborohydride (STAB).

IV. OLIGOSACCHARIDE-PROTEIN CONJUGATES

The invention further provides an oligosaccharide-protein conjugate,comprising (1) a protein, (2) an oligosaccharide, and (3) an oxime groupconnecting the protein and the oligosaccharide. In some embodiments, theinvention provides an oligosaccharide-protein conjugate prepared by themethods disclosed above. The oligosaccharide and protein components ofthe conjugate may be, for example, any oligosaccharide and proteindescribed herein, wherein a conjugate thereof comprises an oxime group,as depicted below. (The oxime group depicted below is formally derivedby reaction of an aminooxy group and an aldehyde group; oxime groupsformally derived by reaction of an aminooxy group and a ketone group arealso encompassed by this invention.)

In certain embodiments, the oligosaccharide-protein conjugate is anoligosaccharide-glycoprotein conjugate. In certain embodiments, theoligosaccharide-protein conjugate is the conjugate of an oligosaccharidecomprising at least one M6P and of a lysosomal hydrolase.

V. PHARMACEUTICAL COMPOSITIONS

This disclosure provides the use of a conjugate of the invention in themanufacture of a medicament for treating a lysosomal storage disorder.It also provides pharmaceutical compositions comprising anoligosaccharide-protein conjugate of the invention. In some embodiments,the pharmaceutical compositions of the invention comprise a conjugate ofan oligosaccharide comprising at least one M6P and a lysosomal enzyme.

Pharmaceutical compositions of the invention may comprise one or moresuitable pharmaceutical excipients. Standard pharmaceutical formulationtechniques and excipients are well known to persons skilled in the art(see, e.g., 2005 Physicians' Desk Reference®, Thomson Healthcare:Montvale, N.J., 2004; Remington: The Science and Practice of Pharmacy,20th ed., Gennado et al., Eds. Lippincott Williams & Wilkins:Philadelphia, Pa., 2000. The compositions may or may not containpreservatives. In some embodiments, pharmaceutical compositionscomprising α-galactosidase A conjugates may comprise one or moreexcipients such as, e.g., mannitol, sodium phosphate monobasicmonohydrate, and/or sodium phosphate dibasic heptahydrate. In someembodiments, pharmaceutical compositions comprising conjugates ofα-glucosidase may comprise one or more excipients such as, e.g.,mannitol, polysorbate 80, sodium phosphate dibasic heptahydrate, andsodium phosphate monobasic monhydrate.

The pharmaceutical composition may comprise any of the conjugatesdescribed herein either as the sole active compound or in combinationwith another compound, composition, or biological material. For example,the pharmaceutical composition may also comprise one or more smallmolecules useful for the treatment of a LSD and/or a side effectassociated with the LSD. In some embodiments, the composition maycomprise miglustat and/or one or more compounds described in, e.g., U.S.Patent Application Publication Nos. 2003/0050299, 2003/0153768;2005/0222244; 2005/0267094.

The formulation of pharmaceutical compositions may vary depending on theintended route of administrations and other parameters (see, e.g., Roweet al. Handbook of Pharmaceutical Excipients, 4th ed., APhAPublications, 2003.) In some embodiments, the composition may be asterile, non-pyrogenic, white to off-white lyophilized cake or powder tobe administered by intravenous injection upon reconstitution withSterile Water for Injection, USP.

Administration of a pharmaceutical composition of the invention is notlimited to any particular delivery system and may include, withoutlimitation, parenteral (including subcutaneous, intravenous,intracranial, intramedullary, intraarticular, intramuscular,intrathecal, or intraperitoneal injection), transdermal, or oral (forexample, in capsules, suspensions, or tablets). Administration to anindividual may occur in a single dose or in repeat administrations, andin any of a variety of physiologically acceptable salt forms, and/orwith an acceptable pharmaceutical carrier and/or additive as part of apharmaceutical composition.

The conjugates described herein are administered in therapeuticallyeffective amounts. Generally, a therapeutically effective amount mayvary with the subject's age, condition, and sex, as well as the severityof the medical condition in the subject. The dosage may be determined bya physician and adjusted, as necessary, to suit observed effects of thetreatment. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in vitro (i.e., cellcultures) or in vivo (i.e., experimental animal models), e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex (or therapeutic ratio), and can be expressed as the ratioLD₅₀/ED₅₀. Conjugates that exhibit therapeutic indices of at least 1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 20 are described herein. Conjugatesthat exhibit a large therapeutic index are preferred.

The data obtained from in vitro assays and animal studies, for example,can be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with low, little, or no toxicity.The dosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any conjugateused in the present invention, the therapeutically effective dose can beestimated initially from in vitro assays. A dose may be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ (i.e., the concentration of the test conjugate whichachieves a half-maximal inhibition of symptoms) as determined in invitro experiments. Levels in plasma may be measured, for example, byhigh performance liquid chromatography or by an appropriate enzymaticactivity assay. The effects of any particular dosage can be monitored bya suitable bioassay of endpoints.

Unless otherwise indicated, conjugates of the invention may beadministered at a dose of approximately from 1 μg/kg to 500 mg/kg,depending on the severity of the symptoms and the progression of thedisease. For example, proteinaceous compounds may be administered byslow intravenous infusion in an outpatient setting every, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more days, or by, e.g., weekly, biweekly,monthly, or bimonthly administration. The appropriate therapeuticallyeffective dose of a compound is selected by a treating clinician andwould range approximately from 1 μg/kg to 500 mg/kg, from 1 μg/kg to 10mg/kg, from 1 μg/kg to 1 mg/kg, from 10 μg/kg to 1 mg/kg, from 10 μg/kgto 100 μg/kg, from 100 μg to 1 mg/kg, and from 500 μg/kg to 5 mg/kg.

For example, conjugates of α-galactosidase A may be administered byintravenous infusion at a dose of, e.g., 1.0 mg/kg body weight every twoweeks at an infusion rate of, e.g., less than or equal to 10, 13, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or33 mg/hour). In another example, conjugates of α-glucosidase may beadministered intravenous injection at a dose of, e.g., 20 mg/kg or 40mg/kg every two weeks, over approximately, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 hours. In some embodiments, the rate of administration ofα-glucosidase may be started at, e.g., 1 mg/kg/hr and then increased by,e.g., 2 mg/kg/hr every 30 minutes, after establishing patient toleranceto the infusion rate, until a maximum of, e.g., 7 mg/kg/hr.Additionally, examples of specific dosages may be found in thePhysicians' Desk Reference®.

VI. METHODS OF TREATING LYSOSOMAL STORAGE DISORDERS

The invention provides methods of treating lysosomal storage disorders,such as, e.g., those disclosed in Table 1. In some embodiments, theinvention provides the use of a conjugate described herein for treatinga lysosomal storage disorder in a subject in need thereof. The inventionfurther provides methods of targeting proteins to the lysosome byconjugation with oligosaccharides comprising mannose-6-phosphate.

In one embodiment, the method comprises administering to a mammal havinga lysosomal storage disorder an oligosaccharide-glycoprotein conjugateof the invention in a therapeutically effective amount. Theoligosaccharide-glycoprotein conjugate may be a conjugate of a lysosomalenzyme, such as a lysosomal enzyme listed in Table 1, with anoligosaccharide comprising mannose-6-phosphate. In one embodiment, themethod comprises administering to a subject in need thereof apharmaceutical composition comprising at least one of the conjugatesdescribed herein.

In certain embodiments, conjugates of the invention may be administeredwith one or more other therapies. The one or more other therapies may beadministered concurrently with (including concurrent administration as acombined formulation), before, or after the administration of theconjugates of the invention.

In some embodiments, a patient may be treated (before, after, or duringtreatment with a conjugate of the invention) with an antipyretic,antihistamine, and/or immunosuppressant. In some embodiments, a patientmay be treated with an antipyretic, antihistamine, and/orimmunosuppressant prior to treatment with anoligosaccharide-glycoprotein conjugate of the invention in order todecrease or prevent infusion associated reactions. For example, patientsmay be pretreated with one or more of acetaminophen, azathioprine,cyclophosphamide, cyclosporin A, methotrexate, mycophenolate mofetil,oral steroids, or rapamycin.

In some embodiments, patients may be treated with one or more ofacetaminophen, azathioprine, cyclophosphamide, cyclosporin A,methotrexate, mycophenolate mofetil, oral steroids, or rapamycin at orabout, e.g., t=0 (the time of administration of the conjugate of theinvention) and/or t=12, 24, 36, 48, 60, 72, 96, 120, and 144 hours for,e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more incidences oftreatment with a conjugate of the invention. For example, in someembodiments a patient with Fabry disease or Pompe disease may be treatedwith methotrexate (e.g., with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 25, 30, 35, 40, 50, 60, 70, 80mg/kg methotrexate, or more) at or about, e.g., t=0, 24, and 48 hoursfor, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 weeks of treatment with aconjugate of the invention. In some embodiments, immune tolerance towardconjugates of the invention may be induced in a patient with a lysosomalstorage disorder such as, e.g., mucopolysaccharidosis I, by treatmentwith cyclosporin A and azathioprine. For example, the patient may betreated with cyclosporine A and azathioprine as described in Kakkis etal., Proc. Natl. Acad. Sci. U.S.A. 101:829-834 (2004).

In some embodiments, a patient may be treated (before, after, or duringtreatment with a conjugate of the invention) with e.g., small moleculetherapy and/or gene therapy, including small molecule therapy and genetherapy directed toward treatment of a lysosomal storage disorder. Smallmolecule therapy may comprise administration of one or more compoundsdescribed in, e.g., U.S. Patent Application Publication Nos.2003/0050299, 2003/0153768; 2005/0222244; and 2005/0267094. Gene therapymay be performed as described in, e.g., U.S. Pat. Nos. 5,952,516;6,066,626; 6,071,890; and 6,287,857 and U.S. Patent ApplicationPublication No. 2003/0087868.

The terms “treatment,” “therapeutic method,” and their cognates refer toboth therapeutic treatment and prophylactic/preventative measures. Thus,those in need of treatment may include individuals already having aparticular lysosomal storage disease as well as those at risk for thedisease (i.e., those who are likely to ultimately acquire the disorderor certain symptoms of the disorder).

A therapeutic method results in the prevention or amelioration ofsymptoms or an otherwise desired biological outcome, and may beevaluated by improved clinical signs or delayed onset of disease,increased activity of the metabolically defective enzyme, and/ordecreased levels of the accumulated substrate of the metabolicallydefective enzyme.

The conjugates of the present invention are useful to treat variouslysosomal storage disorders in humans or animals. For example,administration of the conjugates can be used to increase the deficientenzymatic activity in a patient, for example, by at least 10%. Theincreased enzymatic activity may be determined by, e.g., a reduction inclinical symptoms or by an appropriate clinical or biological assay.

GAA conjugates may be administered for the treatment of Pompe disease(also known as acid α-glucosidase deficiency, acid maltase deficiency,glycogen storage disease type II, glycogenosis II, and lysosomalα-glucosidase deficiency). Increased GAA activity may be determined bybiochemical (see, e.g., Zhu et al., J. Biol. Chem. 279: 50336-50341(2004)) or histological observation of reduced lysosomal glycogenaccumulation in, e.g., cardiac myocytes, skeletal myocytes, or skinfibroblasts. GAA activity may also be assayed in, e.g., a muscle biopsysample, in cultured skin fibroblasts, in lymphocytes, and in dried bloodspots. Dried blood spot assays are described in e.g., Umpathysivam etal., Clin. Chem. 47:1378-1383 (2001) and Li et al., Clin. Chem.50:1785-1796 (2004). Treatment of Pompe disease may also be assessed by,e.g., serum levels of creatinine kinase, gains in motor function (e.g.,as assessed by the Alberta Infant Motor Scale), changes in leftventricular mass index as measured by echocardiogram, and cardiacelectrical activity, as measured by electrocardiogram. Administration ofGAA conjugates may result in a reduction in one or more symptoms ofPompe disease such as cardiomegaly, cardiomyopathy, daytimesomnolescence, exertional dyspnea, failure to thrive, feedingdifficulties, “floppiness,” gait abnormalities, headaches, hypotonia,organomegaly (e.g., enlargement of heart, tongue, liver), lordosis, lossof balance, lower back pain, morning headaches, muscle weakness,respiratory insufficiency, scapular winging, scoliosis, reduced deeptendon reflexes, sleep apnea, susceptibility to respiratory infections,and vomiting.

In another aspect, conjugates of α-galactosidase A with oligosaccharidescomprising M6P are administered for the treatment of Fabry disease.Fabry disease, or Anderson-Fabry disease, is a rare, X-linked, lysosomalstorage disorder marked by a deficiency of α-galactosidase A, andresults in accumulation of globotriaosylceramide (GL3) and other neutralglycosphingolipids in the lysosomes of visceral tissues and endothelial,perithelial, and muscle cells. Accumulation of the neutralglycosphingolipids in the vasculature results in narrowing anddilatation of the blood vessels, and ultimately to ischemia andinfarction.

Administration of α-galactosidase A conjugates may result in a reductionin one or more clinical symptoms of Fabry disease including, e.g.,acroparesthesia, angina, angiokeratoma, arrythmia, ataxia of gait,burning and/or tingling pain in the hands and feet, cataracts, coldintolerance, conduction abnormalities, corneal whorling, coronary arterydisease, dementia, depression, diarrhea, dilated cardiac chambers,dizziness, cardiomegaly, cardiomyopathy, diplopia, dysarthria, fatigue,fever with elevated erythrocyte sedimentation rate, hearing problems,heart disease, heart valve problems, heat intolerance, hemiataxia,hemiparesis, hypohidrosis, impaired sweating, infaraction, ischemia,joint pain, kidney disease, left ventricular hypertrophy, lenticularabnormalities, lenticular opacity, lipiduria, muscle weakness,myocardial infarction, nausea, nystagmus, pain (e.g., intense painradiating throughout the body), polydipsia, proteinuria, post-prandialpain, renal failure, retinal abnormalities, ringing in ears, stomachpain, ST-T wave changes, stroke, uremia, valvular disease, vertigo,vomiting, and weakness. Administration of α-galactosidase A conjugatesmay result in increased α-galactosidase A activity in, e.g., plasma,tears, leukocytes, biopsied tissues, or cultured skin fibroblasts.Administration of α-galactosidase A conjugates may also result in ahistologic finding of a reduction (e.g., of at least 10%) or lack ofincrease of birefringent lipid globules. It may also result in adecrease in lipid globules in urinary sediment, improved renal functionas measured by serum creatinine levels or creatinine clearance, andreduced proteinuria. Administration of α-galactosidase A conjugates mayalso result in a reduction in GL3 inclusions in the capillaryendothelium of the kidney, heart, and skin. Additional assays formeasuring efficacy of treatment for Fabry disease can be found in, e.g.,MacDermott et al., J. Med. Genet. 38:750-760 (2001).

In yet another aspect, conjugates of acid sphingomyelinase areadministered for treatment of Niemann-Pick disease, or acidsphingomyelinase deficiency. Administration of acid sphingomyelinaseconjugates may result in a reduction in one or more clinical symptoms ofNiemann-Pick disease including, e.g., abnormal cholesterol levels,abnormal lipid levels, ataxia, blood abnormalities, cherry red spots inthe eye, frequent lung infections, growth retardation,hepatosplenomegaly, low numbers of platelets, lymphadenopathy,peripheral neuropathy, problems with lung function, shortness of breath,skin pigmentation changes, or xanthomas. In some embodiments, conjugatesmay be administered intracranially.

An alternative embodiment relates to treatment of mucopolysaccharidosisI (including, e.g., Hurler and Hurler-Scheie forms of MPS I) withconjugates comprising α-L-iduronidase. Administration of α-L-iduronidaseconjugates may result in a reduction in one or more clinical symptoms ofMPS I including, e.g., aortic regurgitation, aortic stenosis, carpaltunnel syndrome, chronic rhinitis, conductive hearing loss,constipation, corneal clouding, developmental delay, diarrhea, distendedabdomen, dorsolumbar kyphosis, gibbus deformity of the back,hepatosplenomegaly, hydrocephalus, inguinal hernia, kyphosis, mentalretardation, mitral regurgitation, mitral stenosis, night-blindness,open-angle glaucoma, poor hand function, progressive arthropathy,recurrent respiratory infections, respiratory insufficiency, retinaldegeneration, scoliosis, sensorineural hearing loss, severe back pain,rhinorrhea, sleep apnea, spinal cord compression, thenar atrophy,umbilical hernia, and upper airway complications.

The foregoing and the following description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

EXAMPLES

Examples 1-4 below describe the synthetic route depicted in FIG. 1.Compounds 1, 2, 3, and 4, as used below, have the chemical structuresdepicted in FIG. 1.

Example 1 Synthesis of Oligosaccharide 3

100 mg of oligosaccharide 1 (MW=1250; bisM6P-hydrazide, supplied byBiomira Inc., Edmonton, Canada) was dissolved in 15 ml of DMSO/H₂O(50:50 in volume), yielding a 5.3 μmol/ml solution. 100 mg oft-Boc-aminooxy acetic acid tetrafluorophenyl ester 2 (Invitrogen Corp.;Carlsbad, Calif.; catalog # B3030) was dissolved in 7.5 ml of DMSO. 15ml of the oligosaccharide solution was then mixed with 7.5 ml of thesolution of 2 in a glass bottle, such that the molar ratio of compound2:compound 1 in the resulting solution was 4:1. 744 μl of DHBt-OH (froma 32.06 mg/ml stock in DMSO) was added to the reaction mixture in aglass bottle, such that the final ratio of compound 2:DHBt-OH is 1:0.5.The mixture was gently shaken at room temperature (25° C.) at 100 RPMovernight for about 18 hours.

The following morning, 10 μl of the reaction mixture was removed forDionex analysis to confirm completion of the reaction. The results,depicted in FIG. 2, indicated 100% conversion from 1 to 3.

Example 2 Purification of Oligosaccharide 3

Method A.

The reaction mixture was diluted with an equal volume of H₂O anddialyzed in dialysis tubing with molecular weight cutoff of 1000 Dalton(SpectraPor Inc.) twice against 4 L of H₂O at 4° C. for at least 3 hourseach. The samples were then lyophilized.

Method B.

A Sephadex G-10 gel permeation chromatography column with a bed volumeof 225 ml was packed and equilibrated with deionized water. The reactionmixture was loaded onto the column, drained by gravity, and then elutedwith deionized water at a flow rate of 75 ml per hour. 4.5 ml fractionswere collected with a fraction collector. Fractions 10-23, whichcontained oligosaccharide 3, were collected, combined and lyophilized.The other small molecules, including t-Boc-AOAA, DHBt-OH, and DMSO,eluted in the later fractions, and were discarded.

Example 3 Deprotection of Oligosaccharide 3

The t-Boc group of the lyophilized sample was deprotected in 5 ml of 50%trifluoroacetic acid (TFA) in dicholormethane (DCM) in a glass bottlefor 30 min with gentle shaking at 100 RPM. The TFA/DCM was then removedby a stream of N₂ in a chemical hood.

Example 4 Purification of Oligosaccharide 4

Method A.

After removing the TFA/DCM, the residue was dissolved in 10 ml of 0.5 Msodium acetate buffer, pH 5, and transferred to dialysis tubing with amolecular weight cutoff of 1000 Dalton. The bottle was washed with 4 mlof the same buffer, which was then transferred to the dialysis tubing.The sample was dialyzed twice against 3 L of 25 mM sodium acetatebuffer, pH 7, for at least 3 hours, and then transferred to 4 L ice-coldH₂O for overnight dialysis. The sample was recovered from the dialysistubing and lyophilized.

Method B.

After removing the TFA/DCM, the residue was dissolved in 5 ml of 0.5 Msodium acetate buffer, pH 7.5, and loaded onto a Sephadex G-10 gelpermeation chromatography column as in Example 2, Method B. The reactionmixture was loaded onto the column, drained by gravity, and then elutedwith deionized water at a flow rate of 75 ml per hour. 4.5 ml fractionswere collected with a fraction collector. Fractions 10-23, whichcontained purified oligosaccharide 4, were collected and lyophilized. Ahigher yield of oligosaccharide 4 was obtained upon purification byMethod B than by Method A.

The final product obtained either from method B was analyzed by Dionexchromatography (FIG. 2C), and the identity of the product was confirmedby mass spectrometry (FIG. 3B). Some impurities were present in thespectra of FIG. 2C and FIG. 3B.

Example 5 Coupling of Oligosaccharide 4 to GAA

Oxidation of GAA.

Lyophilized recombinant human GAA (rhGAA) was reconstituted in H₂O anddialyzed against 4 L of 100 mM acetate buffer (pH 5.6) 4 times tocompletely remove mannitol. After dialysis, the rhGAA was oxidized with7.5 mM sodium periodate from 100 mM stock in 100 mM acetate buffer.After 30 minutes at 4° C. on ice, glycerol was added, and the sample wasmixed on ice for 10 minutes to decompose excess sodium periodate. Theoxidized material was then dialyzed against aqueous buffer (e.g., 100 mMsodium acetate) overnight.

Coupling.

A solution of oligosaccharide 4 in aqueous buffer (e.g., 100 mM sodiumacetate, pH 5.6) was mixed with oxidized GAA and incubated at 37° C. for4 hours to yield oligosaccharide-GAA conjugate 5. The reaction mixturewas then diafiltered against 25 mM sodium phosphate buffer, pH 6.25, toremove unconjugated bisM6P glycan, and then adjusted with GAAformulation buffer (25 mM sodium phosphate buffer, pH 6.25, 2% mannitol,0.005% Tween-80).

Example 6 Characterization of the GAA Conjugate

Detection of M6P.

The extent of oligosaccharide conjugation was measured by assayingconjugate 5 for binding to a M6P receptor column to which glycoproteinslacking M6P do not bind. Five micrograms of conjugate 5 were loaded ontoa pre-equilibrated CI-MPR-Sepharose column (the column was prepared bycoupling CI-MPR isolated from fetal bovine serum to Affigel-10), whichwas then washed with CI-MPR binding buffer for 11×2 mL fractions andeluted with CI-MPR binding buffer containing 5 mM M6P for 7×2 mLfractions. A total of 18 fractions were collected and assayed forenzymatic activity.

Monosaccharide Analysis.

Conjugate 5 is treated with 4N trifluoroacetic acid to hydrolyze theoligosaccharides, followed by high pH anion exchange chromatography withpulsed amperometric detection (PAD) on a BioLC liquid chromatographysystem (Dionex). The monosaccharide content is extrapolated from amonosaccharide standard curve using premixed monosaccharide standards(Dionex).

Specific Activity.

GAA activity is measured using a fluorometric assay in black 96-wellmicroplates using 4-methylumbelliferyl-α-D-glucoside as a substrate.Dilutions of conjugate 5 are added in triplicate to a microtiter plate.4-methylumbelliferyl-α-D-glucoside is added to each sample. The 96-wellplate is incubated in a 37° C. incubator for 30 minutes. The release ofproduct is detected fluorometrically, and compared to standard curvesgenerated by measuring the fluorescence of a known quantity of astandard. The reaction is quenched by the addition of 125 μL of 1.0 Mglycine-carbonate buffer, pH 10.5 to all wells. The specific activity isdefined as nmol product released/hr/mg.

Internalization by L6 Myoblasts.

Cells (ATCC CRL-1458) were seeded into 6-well plates at 5.0×10⁵cells/well in growth media (DMEM+10% FBS) and grown to confluency. Cellswere incubated with 0-100 nM GAA (conjugate 5 or unconjugated rhGAA) for16 hours in DMEM+1% heat-inactivated-FBS+10 mM Hepes pH 6.7. Afteruptake, cells were washed with 3×PBS containing 5 mM M6P and lysed with0.25% Triton X-100 for 1 hour on ice. Lysates were centrifuged at 18000g for 5 minutes and tested for specific activity. See, e.g., Zhu et al.,J. Biol. Chem. 279:50336-50341 (2004); Zhu et al., Biochem. J.389:619-628 (2005).

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the inventioncontained in the specification, the specification is intended tosupercede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1-55. (canceled)
 56. A method of coupling an oligosaccharide to aprotein, comprising: (a) providing an oligosaccharide comprising anaminooxy group; (b) providing a protein having at least one carbonylgroup; and (c) reacting the aminooxy group of the oligosaccharide withthe at least one carbonyl group of the protein, thereby coupling theoligosaccharide to the protein, wherein the oligosaccharide comprisingan aminooxy group is

wherein m is an integer from 1 to 10, and p is an integer from 1 to 10.57. The method of claim 56, wherein the protein is a glycoprotein andthe at least one carbonyl group is obtained by oxidation of theglycoprotein with periodate.
 58. The method of claim 56, wherein theprotein is a lysosomal enzyme.
 59. The method of claim 58, wherein thelysosomal enzyme is acid α-glucosidase, α-galactosidase A, acidsphinogyelinase, or α-L-iduronidase.
 60. The method of claim 58, whereinthe lysosomal enzyme is acid α-glucosidase.
 61. The method of claim 56,wherein m is
 3. 62. The method of claim 56, wherein p is
 1. 63. Themethod of claim 56, wherein m is 3 and p is
 1. 64. The method of claim63, wherein the protein is acid α-glucosidase.
 65. Anoligosaccharide-protein conjugate produced by the method of claim 56.66. A pharmaceutical composition comprising the oligosaccharide-proteinconjugate of claim 65 and an excipient.